1. Field of the Invention (Technical Field)
This invention relates generally to the measurement of organisms and free antigens present or suspended in water by the detection of selected antigen-antibody reactions and more specifically to both a hand-held and an on-line sensor for monitoring the concentration of organisms and free antigens.
2. Background Art
The detection of both organisms (i.e., bacteria, fungi, protozoans, algae, and viruses, both pathogenic and non-pathogenic) and selected organic pollutants in environmental fluid streams or in industrial process streams, including waste effluents, is becoming extremely important. Several prior art techniques exist for monitoring the concentration of organisms or selected organic pollutants in fluids, but each of these methods has several drawbacks in either environmental aqueous fluids or industrial process streams, including waste effluents in either a field or on-line industrial setting.
Standard microbiological procedures for detecting specific microbes and quantifying them includes several processes, all being time consuming and expensive.
One microbiological technique involves the drawing of a sample aliquot from the contaminated waters and the streaking of standard dilutions of that sample aliquot onto a selective agar growth medium which promotes the growth of the organism of interest. The streaked plates then are incubated under optimum growth conditions (temperature, pH, aeration, etc.) for the organism of interest for a period of time (usually 24 to 48 hours). The growth colonies appearing on the selective medium after the appropriate time period often must be examined under the microscope using staining techniques. This process often will yield related family or genera members, not a specific species or strain of a species. Often, the colonies of interest must be further subcultured one or more times on additional specific growth media in order to obtain definitive speciation or strain selection. Unless one is lucky with the first growth (i.e., showing countable, uniquely identifiable colonies), no quantification of species or strain can be made by this process, only the presence of family or genera members can be deduced.
Speciation or strain specificity techniques consist of mixing suspensions of specific colonies (obtained from the selective growth media, as described above) with specific polyclonal or monoclonal antibodies and observing the results of the formation of the antigen-antibody complex. Antigen-antibody agglutination complex formation reactions often can be seen by the naked eye. Fluorescent antigen-antibody complexes may be seen only by the use of a special fluorescent microscope. These tests, too, are usually qualitative. However, by making complicated standard dilutions and subsequent tests, lower threshold values for the different antigen-antibody complex formations may be determined for pure cultures. Newer techniques, using monoclonal-antibodies coated with colloidal gold, can provide specific and semi-quantitative estimations for selected microbes. At this time, even though the test specificity is very high, it is unknown whether these tests would be applicable to the rapid detection and enumeration of specific environmental water samples.
Detection of selected organic molecules using immunoassay techniques is relatively new. One new technique being used for selected organic molecules of interest is a haptenization technique. A hapten is a small molecule that is incapable of eliciting an antibody formation by itself, but can serve as a partial antigen if it is attached to the antigenic determinant of the larger, antigenic, macromolecule. The selected organic molecules of interest are haptenized onto the antigenic determinants of a selected macromolecule (antigen). An antibody is formed against the haptenized macromolecule by the injection of the haptenized molecule into a certain animal. A hapten-enzyme conjugate will mimic free organic molecules of interest and will compete for binding to the polyclonal antibody immobilized on a test tube. After washing to remove the unbound conjugate, a substrate chromogen is added and a colorized enzymatic reaction product is formed. The enzymatic reaction is stopped by the addition of a few drops of sulfuric acid, which changes the color to yellow. As with other competitive, enzyme-linked immunosorbent assays (ELISA), the color intensity of the enzymatic product is inversely proportional to the sample analyte concentration. Each sample must be run with a reference sample of deionized water. The optical density of the colored enzymatic product is read on a colorimeter or spectrophotometer. The ratio of the sample to the reference optical density values is used to estimate the selected organic chemical level, usually sensitive to the low parts per million (milligrams per liter) range. At this time, the technique seems to be restricted to estimating the level of polycyclic aromatic hydrocarbons in waters.
A more common method for determining the presence of selected organic molecules consists of collecting and measuring the molecules of interest by the use of one, or a combination of several, complicated techniques. These techniques include gas chromatography (GC), mass spectrometry (MS), high pressure liquid chromatography (HPLC), or combined techniques or instrumentation (e.g., GC-MS, HPLC-MS, etc.). In some cases, gas chromatography or liquid column chromatography used in conjunction with photometric detection has been used for organic substances which either absorb light or can be made to absorb light.
In each of the above methods of gas or liquid chromatography, the column uses a separation means for separating and individually detecting the molecules of interest. These column gas or liquid chromatography methods are not practical for either field or on-line sensors and the photometric measurements are subject to interference between different organic species contained in the sample which have similar retention times in the chromatographic column. Further, these methods require the injection of a sample and then waiting for the chromatographic column to separate the different species in time so that the concentration of each organic species in the sample can be determined by photometrically or electrically monitoring the effluent from the column. None of these methods would function adequately if a continuous stream sample were provided to the chromatographic columns.
In another method using chromatographic columns, Proudfoot, in U.S. Pat. No. 4,396,718 and U.S. Pat. No. 4,268,269, teaches a two step method for the detection of triazoles in an aqueous solution. A separation step removes the triazoles from the aqueous solution by adsorption onto a molecular resin. Next, an eluting solvent is passed through the molecular resin containing the previously adsorbed triazole. The eluting solvent desorbs the triazoles, and the eluate from the column is essentially free from impurities which would interfere with the quantification of the amount of triazole. In the quantification step, the eluate containing the triazole is passed through a column containing a cation exchange resin to which is bound a metal ion, wherein the triazole is strongly bound to the surface of the resin as a colored metal ion-triazole complex. After removal of uncomplexed metal from the column, the size and color of complexed metal-triazole bands formed on the column are visually compared with a known standard to determine the concentration of triazoles. This method is impractical for field or on-line monitoring because a visual measurement is necessary for quantification. Further, since the triazole is strongly bound to the surface of the resin as a colored metal iontriazole complex, the triazoles cannot be easily stripped from the column and the column reused for the next measurement.
Other methods use colorimetric techniques to detect organic molecules in an aqueous solution. For example, Rothman et al., U.S. Pat. No. 4,652,530, teaches a method for a colorimetric determination of isothiazolones in a fluid stream. The isothiazolones are first concentrated on a nonpolar adsorbent. The isothiazolones are stripped from the adsorbent and a reagent is used to break the aromatic ring of the isothiazolones. Then, another reagent is added to produce a colored complex. A conventional colorimetric analysis of the colored complex is used to determine the concentration of the isothiazolone.
Laman et al., U.S. Pat. No. 3,558,277, teaches a method for detecting biodegradable organics in aqueous solution in which the fluid stream is first mixed with a material to precipitate metals from the stream. The stream is filtered to remove the precipitate and then mixed with a permanganate solution and heated for 30-40 minutes. The solution is then diluted and colorimetrically analyzed.
Automated colorimetric methods rely on an expensive colorimeter to analyze the final product. The colorimetric equipment is not a compact self-contained unit that is easily installed in an on-line industrial setting.
Bedell et al., U.S. Pat. No. 4,908,676, discloses an on-line analyte detection system (analyte detector) that detects both selected inorganic and organic analytes of interest which are dissolved in a process stream. A side stream is extracted through a motor-driven valve from the process stream, either by pressure relief or by pumping. The side stream flows through a detection column of an analyte detector and a sensor mounted around the detection column which detects the analyte by use of electromagnetic radiation and a sensor of that radiation. The side stream leaves the detection column and enters the line where the stream flows through a second motor-operated valve and then returns to the main process stream.
The sensor contains a source of electromagnetic radiation, typically a light source from a light emitting diode (LED), and an electromagnetic radiation detector, typically a Darlington phototransistor, and a detection that is transparent to the electromagnetic radiation source, typically a glass, plastic, or quartz tube. In one embodiment, the LED and the light detector are contained 180.degree. apart in the sensor. Hence, the detector measures the light from the LED transmitted through the contents of the detection column. In another embodiment, the detector is located in the sensor so that the light from the LED is reflected by the contents of the detection column into the light detector. In yet another embodiment, the LED and the light detector are located at approximately 60.degree. to 90.degree. to each other such that the LED causes the contents of the detection column to fluoresce and the light detector (shielded from the incident light by the appropriate cut-off or interference filter) measures the fluorescent light.
Accordingly, the optical properties of the analyte and/or the adsorbed analyte define the configuration of the sensor that is used in the inline detector. If the analyte in the process stream has electromagnetic radiation absorption, scattering, transmission, or emission properties that can be detected by the sensor, the side stream is simply passed through the detection column and the electromagnetic radiation properties are detected by the sensor.
If the analyte does not have the necessary optical properties, or if it is desired to measure the total amount of analyte in the process stream in a given period, the detection is packed with an adsorbent so that as the side stream from the main line flows through the detection column, the adsorption of the analyte species of interest in the side stream flow forms a complex on the adsorbent, removes the analyte from the side stream and effectively concentrates the analyte so that its presence is easily detected by the sensor. Also, the complex formed on the adsorbent may have a significantly different extinction coefficient from the analyte itself. Accordingly, the formation of the complex on the adsorbent enhances the capability of detecting the presence of the analyte in the process stream.
A controller is used to sequence the operation of the on-line analyte detector. Initially, the controller holds closed the motor-operated valve to a first reagent tank, the motor-operated valve to a second reagent tank containing an eluent, and the motor-operated valve to the waste recovery system. The controller activates a timer, which determines the sampling period, i.e., the prescribed time interval, of the analyte detector, and opens the valves so that the side stream starts to flow though the on-line analyte detector. While motor-operated valves are used in this embodiment, any valve, which can be remotely controlled, can be used in the system.
In response to the detector sensor function, the sensor circuit energizes the alarm to alert personnel of the process stream concentration or deviations from some predetermined value and energizes a circuit in the controller which takes the analyte detector off-line prior to the end of the sampling period, etc. The controller then aligns the eluent reagent tank with the detection column for stripping.
Some analytes do not have an absorption or emission spectrum which is detectable, and other analytes are not conveniently adsorbed on any matrix even though the analyte has acceptable electromagnetic spectral characteristics. In these cases, the analyte detector utilizes either a reaction product of the analyte wherein the reaction product has acceptable optical properties, or the reaction product is better adsorbed on the adsorbent in the detection column. In these instances, the controller is set so that a motor-operated valve to one of the reagent tanks and the reagent in the reagent tank is metered into the side stream in line before entry into the detector column. The interaction of the reagent with the analyte in the side stream produces a product which has detectable spectral properties and/or which is adsorbed on the adsorbent in the detection column. The sensor is set as previously described, and the operation of the analyte detector also is as previously described.
The analyte detectors are placed in an opaque container to prevent external electromagnetic radiation from affecting the readings of the photodetector contained within the sensor. The patent of Bedell et al., also describes an embodiment in which both the electromagnetic source and the sensor are mounted outside of the body of the sensor. In this embodiment, a first fiber optic cable is secured at the position of the incident source, typically the LED, and the second fiber optic is secured at the position of the sensor, typically the phototransistor position on the analyte detector collar. The cables are secured in the holes so that they are flush with the inner surface of the sensor body formed by the hole for the detection column. One of the fiber optic cables is connected to a light source (LED, laser, etc.) outside the sensor body and the other fiber optic cable is connected to the photodetector which also now is outside the sensor body. This embodiment permits the use of larger, and in certain cases, more sensitive, light sources and photodetectors.
Each of the prior art systems suffers from several deficiencies which make the field and on-line detection of microorganisms and selected organic molecules in aqueous solution impractical, particularly from the points of specificity, time, and expense. Microbiological techniques are especially time consuming and expensive, with specific immunoassay procedures generally being followed as adjuncts to the original isolation techniques. Also, the latter techniques which may have field applications generally require the presence of large numbers of the organism of interest. Current immunoassay techniques are not being used as on-line sensors. Liquid or gas chromatographic and mass spectrographic equipment are both complex and expensive. The chromatographic methods primarily are designed to distinguish multiple ions (organic chemical) and the concentration of each ion (organic chemical). Chromatographic methods generally are not suitable for either continuous monitoring or the monitoring of the amount of ion or organic chemical in a mixed process stream during a specific time period. The process of Bedell et al., does provide an exception for allowing the on-line determination of selected organic molecules, but it does not allow for the detection of microorganisms or the use of immunoassay techniques for determining selected organic analytes.